This revised Program Project renewal application builds on the collaborative relationships and scientific accomplishments of the past funding period in order to continue to advance our understanding of the role of antigen-specific and cross-reactive (XR) CD8+ T cells in the induction and maintenance of memory responses. Project investigators will use the Epstein Barr virus (EBV) model of infection with unique clinical cohorts to: 1) characterize the lineage relationship between effector and memory CD8+ T cell responses in humans and the factors that influence the evolution of antigen-specific CD8+ T cell responses into the memory CD8+ T cell repertoire; 2) characterize how cross-reactivity influences the evolution of the antigen specific CD8+ T cell repertoire and understand the structural and functional properties of the T cell receptor that contribute to the recognition of antigen-specific or XR ligands; 3) correlate antigen-specific and XR CD8+ T cell frequencies and functional properties with control of viral replication, evolution of viral sequences, and manifestations of disease. Project 1 will use TCR CDR3? and ? sequencing and genome wide microarray analyses to characterize the lineage relationship between effector and memory CD8+ T cell responses and the factors that influence evolution of EBV-specific CD8+ T cell responses into the memory CD8+ T cell repertoire. Project 1 will also use novel deep sequencing methods to analyze EBV sequence diversity in the oral and peripheral blood compartments and will examine the potential role of EBV epitope specific CD8+ T cell responses in EBV sequence evolution. Project 2 will further characterize the human XR CD8 T cell repertoire and determine how cross-reactivity impacts memory CD8+ T cell selection and function and correlates with disease outcomes. Three core facilities will facilitate the work of Project Investigators. Core A (Clinical and Administrative) will provide scientific leadership; manage fiscal affairs; collect, process, and disburse clinical specimens from research subjects; and develop and maintain a comprehensive clinical and laboratory database. The Tetramer Core (Core B) will provide MHCI-peptide tetramers to all Project Investigators. Core C (Flow Cytometry) provides access to state-of-the-art facilities for FACS-based cell sorting and analyses of virus-specific or XR CD8+ T cells. The proposed studies will improve our understanding of the role of antigen-specific and XR CD8+ T cells in the induction and long-term maintenance of memory CD8+ T cell responses. These studies will also help us to understand variability in human responses to viral infections and how antigen-specific or XR CD8+ T cells contribute to either viral control or disease. RELEVANCE: Understanding the mechanisms operative in the establishment of persistent viral infections and in the evolution of virus specific and cross-reactive CD8+ T cell responses should contribute to the development of new or improved viral vaccines and the development of novel therapies for autoimmune diseases or virus-associated malignancies. Project 1: Evolution and Maintenance of EBV-specific Memory CD8+ T Cells Project Leader: Katherine Luzuriaga DESCRIPTION (provided by applicant): Program Project #1 will use the EBV model of persistent viral infection to characterize the lineage relationship between effector and memory responses and the factors that influence the evolution of antigen specific CD8+ T cell responses into the memory CD8+ T cell repertoire. Work outlined in this project addresses the collective hypotheses that virus-specific CD8+ T cell repertoires change quantitatively and qualitatively over time following acute infection, and that differences in virus-specific CD8+ T cell repertoires may influence the control of viral replication, viral sequence evolution, and disease pathogenesis. Changes in the EBV-specific CD8+ T cell repertoire over time may be based on inherent properties of EBV-specific CD8+ T cell clones (specificity, TCR usage, avidity, genome-wide transcriptional profiles), prior infections and ongoing antigenic exposure. Specific Aim 1 wil use TCR CDR3? and ? sequencing to define the relationship between the EBV epitope-specific TCR repertoires at serial time points from the acute through the memory phases of the T cell response. Specific Aim 2 will define genome-wide expression profiles in EBV epitope-specific CD8+ T cells during the acute phase of the T cell response and CD8+ T cell persistence into the memory pool in order to examine whether specific gene expression signatures correlate with the size of the EBV epitope-specific CD8+ T cell response during AIM, persistence or non-persistence of the EBV epitope-specific CD8+ T cell response, control of EBV replication, and/or AIM symptoms in primary infection. Specific Aim 3 will use novel deep sequencing methods to define EBV founder populations and track EBV sequence evolution in oropharyngeal secretions and peripheral blood B cells over time following infection. Characterization of intra-host EBV genetic sequence variability should improve our understanding of anatomical sites of EBV replication and persistence and should better define factors (viral replication, repeated infections, immune selective pressures) that contribute to EBV sequence diversity over time. Project 1 integrates experimentally and conceptually with the proposed characterization of cross-reactive CD8+ T cells in Project 2 (Selin) and will use the services of all 3 Cores. RELEVANCE: The proposed studies along with those in Project 2, will provide insight into factors that control the evolution of human antiviral CD8+ T cell responses from the acute effector phase into long-term memory and how antigen-specific or cross-reactive CD8+ T cells may contribute to either viral control or disease. Ultimately, we hope that they will help to infor the development of antiviral vaccines that afford long-term protection against infection or disease, while minimizing the potential adverse effects of these vaccines.